Mixed disulfide conjugates of thienopyridine compounds and uses thereof

ABSTRACT

This invention is in the field of medicinal chemistry. In particular, the invention relates to mixed disulfide conjugates of thienopyridine compounds, and their use as therapeutics for the treatment, amelioration, and prevention of cardiovascular diseases.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AA020090 andCA016954 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention is in the field of medicinal chemistry. In particular,the invention relates to mixed disulfide conjugates of thienopyridinecompounds, and their use as therapeutics for the treatment,amelioration, and prevention of cardiovascular diseases.

INTRODUCTION

Thienopyridinyl compounds are widely used as antiplatelet agents toprevent heart attack and stroke. In this category, clopidogrel (Plavix),ticlopidine (Ticlid) and prasugrel (Effient) are three commonly usedprodrugs. These agents require polymorphic cytochrome (P450) mediatedoxidative bioactivation. Such oxidative bioactivation results in slowon-set of therapeutic effect and several adverse effects includingneutropenia and thrombotic thrombocytopenic purpura.

Improved antiplatelet agents not requiring polymorphic cytochrome (P450)mediated oxidative bioactivation are needed.

SUMMARY OF THE INVENTION

Clopidogrel (Plavix), ticlopidine (Ticlid) and prasugrel (Effient)belong to a class of thienopyridinyl compounds widely used asantiplatelet agents to prevent heart attack and stroke. However, severalserious drawbacks have been associated with these drugs including lackof response, toxicity and excessive bleeding. These drawbacks areclosely related to the fact that they are all prodrugs that requireoxidative bioactivation by polymorphic cytochromes P450 enzymes (P450s).

To overcome drawbacks associated with thienopyridine compounds(Clopidogrel (Plavix), ticlopidine (Ticlid) and prasugrel (Effient)),mixed disulfide conjugates of thienopyridine compounds were developed.Indeed, experiments conducted during the course of developingembodiments for the present invention demonstrated that the mixeddisulfide conjugates of thienopyridine compounds of the presentinvention are capable of producing active thienopyridine metabolites(e.g., active thienopyridine metabolites capable of antiplateletactivity) in the presence of endogenous glutathione (GSH) without theneed for bioactivation by P450s. This approach not only bypasses theoxidative bioactivation process by P450s, but circumvents many of thedrawbacks associated with thienopyridinyl drugs. For example, the mixeddisulfide conjugates of thienopyridine compounds of the presentinvention improve dosing consistency because production of the activemetabolite from the conjugates is predictable. In addition, use of themixed disulfide conjugates of thienopyridine compounds of the presentinvention as antiplatelet agents reduce the toxicity as toxic reactivemetabolites are not produced by the thiol-exchange reaction. Inaddition, the therapeutic onset time for the mixed disulfide conjugatesof thienopyridine compounds of the present invention is shortened, whichgreatly benefits patients who experience acute cardiovascular events.For example, the standard regimen for thienopyridines requirescontinuously dosing patients for 3-5 days as only a small percentage ofingested thienopyridines are converted to the active metabolite. Incontrast, the mixed disulfide conjugates of thienopyridine compounds ofthe present invention release the active metabolites with high yields inless than 30 min. In addition, the mixed disulfide conjugates ofthienopyridine compounds of the present invention have superiorstability over the active metabolites and therefore they can be used toquantitatively generate the active metabolites for basic and clinicalresearch in vitro.

Accordingly, in certain embodiments, the present invention providesmixed disulfide conjugates of thienopyridine compounds capable ofovercoming such drawbacks associated with thienopyridinyl compoundswidely used as antiplatelet agents (e.g., Clopidogrel (Plavix),ticlopidine (Ticlid) and prasugrel (Effient)). The present invention isnot limited to particular mixed disulfide conjugates of thienopyridinecompounds. In some embodiments, the mixed disulfide conjugates ofthienopyridine compounds are described by Formula I:

including pharmaceutically acceptable salts, solvates, and/or prodrugsthereof; wherein R1, R2, and R3 independently include any chemicalmoiety that renders the resulting compound capable of producing activethienopyridine metabolites upon interaction with endogenous glutathione(GSH) (e.g., active thienopyridine metabolites capable of antiplateletactivity).

In some embodiment, R is selected from the group consisting of H,—CO—OCH3, and

In some embodiments, R3 is Chlorine or Fluorine.

In some embodiments, R2 is selected from the group consisting of

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising a mixed disulfide conjugate of a thienopyridinecompound and a pharmaceutically acceptable carrier.

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising mixed disulfide conjugates of thienopyridinecompounds configured for intravenous (IV) administration. In someembodiments, such pharmaceutical compositions comprising mixed disulfideconjugates of thienopyridine compounds configured for intravenous (IV)administration are used in the treatment, amelioration and prevention ofatherothrombosis. In some embodiments, such pharmaceutical compositionscomprising mixed disulfide conjugates of thienopyridine compoundsconfigured for intravenous (IV) administration are used for rapidinhibition of platelet aggregation. In some embodiments, suchpharmaceutical compositions comprising mixed disulfide conjugates ofthienopyridine compounds configured for intravenous (IV) administrationare used during percutaneous coronary intervention procedures (e.g.,coronary angioplasty) for rapid inhibition of platelet aggregation.

In certain embodiments, the present invention provides methods oftreating, ameliorating, or preventing a cardiovascular diseasecomprising administering to a patient a therapeutically effective amountof a mixed disulfide conjugate of a thienopyridine compound. In someembodiments, the administration is intravenous administration. In someembodiments, the cardiovascular disease is selected from the groupconsisting of coronary artery disease, peripheral vascular disease, andcerebrovascular disease. In some embodiments, the compound reducesaggregation of platelets (e.g., through irreversible binding to P2Y₁₂receptors) (e.g., through blocking ADP receptors). In some embodiments,the compound is capable of producing active thienopyridine metabolitesin the presence of endogenous glutathione without the need forbioactivation by P450s. In some embodiments, the methods furthercomprise co-administration of at least one agent selected from the groupconsisting of a HMG-CoA reductase inhibitor, an ACE Inhibitor, a CalciumChannel Blocker, a Platelet Aggregation Inhibitor, a PolyunsaturatedFatty Acid, Fibric Acid Derivative, a Bile Acid Sequestrant, anAntioxidant, and an Antianginal Agent.

In certain embodiments, the present invention provides methods oftreating, ameliorating, or preventing aggregation of platelets ontoblood vessels in a patient, comprising administering to the patient atherapeutically effective amount of a mixed disulfide conjugate of athienopyridine compound. In some embodiments, the administration isintravenous administration. In some embodiments, the patient has or isat risk for developing cardiovascular disease (e.g., coronary arterydisease, peripheral vascular disease, and cerebrovascular disease). Insome embodiments, the treating, ameliorating, or preventing theaggregation of the platelets occurs through irreversible binding toP2Y₁₂ receptors. In some embodiments, the treating, ameliorating, orpreventing the aggregation of the platelets occurs through blocking ADPreceptors. In some embodiments, the mixed disulfide conjugate ofthienopyridine compound is capable of producing active thienopyridinemetabolites in the presence of endogenous glutathione without the needfor bioactivation by P450s.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows effects of thiol reductants on the formation of the activemetabolite (AM) of clopidogrel. The AM was produced in 0.1 ml of 50 mMKP buffer (pH 7.4) containing 0.2 mg/ml HLM, 0.1 mM 2-oxoclopidogrel,the NADPH-regenerating system, and the thiol reductants. Theconcentrations of the thiol reductants were 1 mM except for CPT, DFT andNPT which were 0.3 mM each. The reaction was initiated by the additionof 5 units of G6PD and incubated at 37° C. for 20 min. The activemetabolite was then quantitated as the MP derivative as described inMaterials and Methods. The reported rates were averaged over threeseparate measurements. Abbreviations for the thiol compounds areprovided in Table 1.

FIG. 2 shows extracted ion chromatograms (EIC) of representative mixeddisulfide conjugates of clopidogrel. The mixed disulfide conjugates wereproduced in 0.2 ml of 50 mM KPi buffer (pH 7.4) containing 1 mg/ml HLMs,0.1 mM 2-oxoclopidogrel, various thiol reductants and theNADPH-regenerating system at 37° C. for 30 min. MS analyses wereperformed as described in Materials and Methods. (A), EIC for the BMEconjugate at m/z 432.06; (B) EIC for the DFT conjugate at m/z 482.08;(C) EIC for the CPT conjugate at m/z 499.99; (D) EIC for the NPTconjugate at m/z 510.08.

FIG. 3 shows relative amounts of the AM and thiol conjugates ofclopidogrel produced by HLMs. The AM and conjugates were produced in 0.2ml of 50 mM KP (pH 7.4) as described in FIG. 2. For these analyses, 50pmoles of (S)-clopidogrel was spiked into each sample as the IS. Boththe AM and the thiol conjugates were analyzed using LC-MS/MS in thedependent scan mode as described in Materials and Methods. Legend: openbar, AUC ratio of m/z 356 (AM) to m/z 322 (IS); solid bar, AUC ratios ofrespective conjugate to IS.

FIG. 4 shows MS and MS² spectra of the mixed disulfide conjugate of CPT.The conjugate was produced as described in FIG. 2. The MS and MS²spectra were obtained using LC-MS/MS in the dependent-scan mode asdescribed in Materials and Methods. (A), MS spectrum of the CPTconjugate; (B) MS² spectrum of the parent ion m/z 499.99 for the CPTconjugate; (C), MS' spectrum of the parent ion m/z 501.94 for the CPTconjugate; (D), assignments for the fragmentation pattern shown in FIG.4B.

FIG. 5 shows kinetics for the reduction of the mixed disulfideconjugates of clopidogrel by GSH. The mixed disulfide conjugates wereprepared from the reaction mixtures containing 1 mg/ml HLM, 0.1 mM2-oxoclopidogrel, the NADPH-regenerating system and various thiolreductants and purified using SPE C18 cartridges as described inMaterials and Methods. The purified conjugates were then mixed with 1 mMGSH and 0.2 mg/ml cytosol (when present). The conjugate remaining andthe AM formed were analyzed using LC-MS/MS. Legend: (A) reduction of theconjugates of BME (◯), CPT (▪), NAC (▾), DFT (▴), and NPT (□) by 1 mMGSH in the presence of 0.2 mg/ml cytosol. (B) reduction of the CPTconjugate by 1 mM GSH in the presence and absence of 0.2 mg/ml cytosol.Legend: (◯), formation of AM in the absence of cytosol; (Δ), formationof the AM in the presence of cytosol; (●), reduction of the conjugate inthe absence of cytosol; (▴), reduction of the conjugate in the presenceof cytosol. The solid and dashed lines are non-linear curve fittings toa single exponential function.

FIG. 6 shows extracted ion chromatograms observed at m/z 504 showing theformation of the active metabolite H4 from the mixed disulfideconjugates of clopidogrel. The mixed disulfide conjugates were producedin the HLMs and purified with SPE cartridges as described in FIG. 3.Prior to MS analyses, the mixed disulfide conjugates were treated withDTT to release the AM that was then subsequently derivatized with MPB.The AM-MP derivatives were analyzed using LC-MS/MS as described inMaterials and Methods. Legend: A, trans- (dashed line) andcis-clopidogrel-MP (solid line) standards; B, the AM-MP obtained in thepresence of 1 mM GSH (dashed line) and 1 mM ascorbic acid (solid line);C, the AM-MP obtained from the CPT conjugate; D, the AM-MP obtained fromthe NPT conjugate; E, the AM-MP obtained from the DFT conjugate. Theamplitude was multiplied by two.

FIG. 7 shows inhibition of platelet aggregation by the mixed disulfideconjugates of clopidogrel. The mixed disulfide conjugates were preparedand purified from the reaction mixtures in the presence of 0.3 mM CPTand NPT, along with three control samples containing either 1 mM GSH, nothiol reductant or no G6PD. All samples were re-suspended in 0.5 ml PPP,some of which were treated to 1 mM GSH to release the AM. Plateletaggregation was initiated by the addition of 10 μM ADP and recorded withan aggregometer. The percentage of aggregation was normalized to that ofPRP and averaged over four separate measurements. For details, seeMaterials and Methods. Legend: PRP, untreated platelet-rich plasma; GSH,1 mM GSH in PRP; -G6PD, metabolites produced in the absence of G6PD;—SH, metabolites produced in the absence of any thiol reductants; GSH,metabolites produced in the presence of 1 mM GSH; CPT, metabolitesproduced in the presence of 0.3 mM CPT; CPT+GSH, metabolites produced inthe presence of 0.3 mM CPT and then treated with 1 mM GSH; NPT,metabolites produced in the presence of 0.3 mM NPT; NPT+GSH, metabolitesproduced in the presence of 0.3 mM NPT and then treated with 1 mM GSH.

FIG. 8 presents a total ion chromatogram of pure (S)-clopNPTbio-synthesized in the reconstituted systems as described in Example X.The three diastereomers of (S)-clopNPT were eluted at 7.9, 8.6, and 9.5min.

FIG. 9 presents platelet activities of male NZ white rabbits followingIV injection of (S)-clopNPT as described in Example X.

DEFINITIONS

The term “thienopyridine compound” as used herein, refers to a class ofADP receptor/P2Y12 inhibitors used for their anti-platelet activity.Examples include, but are not limited to, clopidogrel (Plavix),ticlopidine (Ticlid), and Prasugrel (Effient).

The term “mixed disulfide conjugate of a thienopyridine compound” asused herein, refers to a modified thienopyridine compound capable ofproducing active thienopyridine metabolites upon interaction withendogenous glutathione (GSH).

The term “prodrug” as used herein, refers to a pharmacologicallyinactive derivative of a parent “drug” molecule that requiresbiotransformation (e.g., either spontaneous or enzymatic) within thetarget physiological system to release, or to convert (e.g.,enzymatically, physiologically, mechanically, electromagnetically) theprodrug into the active drug. Prodrugs are designed to overcome problemsassociated with stability, water solubility, toxicity, lack ofspecificity, or limited bioavailability. Exemplary prodrugs comprise anactive drug molecule itself and a chemical masking group (e.g., a groupthat reversibly suppresses the activity of the drug). Some prodrugs arevariations or derivatives of compounds that have groups cleavable undermetabolic conditions. Prodrugs can be readily prepared from the parentcompounds using methods known in the art, such as those described in ATextbook of Drug Design and Development, Krogsgaard-Larsen and H.Bundgaard (eds.), Gordon & Breach, 1991, particularly Chapter 5: “Designand Applications of Prodrugs”; Design of Prodrugs, H. Bundgaard (ed.),Elsevier, 1985; Prodrugs: Topical and Ocular Drug Delivery, K. B. Sloan(ed.), Marcel Dekker. 1998; Methods in Enzymology, K. Widder et al.(eds.). Vol. 42, Academic Press, 1985, particularly pp. 309-396;Burger's Medicinal Chemistry and Drug Discovery, 5th Ed., M. Wolff(ed.), John Wiley & Sons, 1995, particularly Vol. 1 and pp. 172-178 andpp. 949-982; Pro-Drugs as Novel Delivery Systems, T. Higuchi and V.Stella (eds.), Am. Chem. Soc., 1975; and Bioreversible Carriers in DrugDesign, E. B. Roche (ed.), Elsevier, 1987.

Exemplary prodrugs become pharmaceutically active in vivo or in vitrowhen they undergo solvolysis under physiological conditions or undergoenzymatic degradation or other biochemical transformation (e.g.,phosphorylation, hydrogenation, dehydrogenation, glycosylation).Prodrugs often offer advantages of water solubility, tissuecompatibility, or delayed release in the mammalian organism. (See e.g.,Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam(1985); and Silverman, The Organic Chemistry of Drug Design and DrugAction, pp. 352-401, Academic Press, San Diego, Calif. (1992)). Commonprodrugs include acid derivatives such as esters prepared by reaction ofparent acids with a suitable alcohol (e.g., a lower alkanol) or estersprepared by reaction of parent alcohol with a suitable carboxylic acid,(e.g., an amino acid), amides prepared by reaction of the parent acidcompound with an amine, basic groups reacted to form an acylated basederivative (e.g., a lower alkylamide), or phosphorus-containingderivatives, e.g., phosphate, phosphonate, and phosphoramidate esters,including cyclic phosphate, phosphonate, and phosphoramidate (see, e.g.,US Patent Application Publication No. US 2007/0249564 A1).

The term “pharmaceutically acceptable salt” as used herein, refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target animal (e.g., a mammal). Salts of the compounds of thepresent invention may be derived from inorganic or organic acids andbases. Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

The term “solvate” as used herein, refers to the physical association ofa compound of the invention with one or more solvent molecules, whetherorganic or inorganic. This physical association often includes hydrogenbonding. In certain instances, the solvate is capable of isolation, forexample, when one or more solvate molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Exemplary solvates includehydrates, ethanolates, and methanolates.

The term “therapeutically effective amount,” as used herein, refers tothat amount of the therapeutic agent sufficient to result inamelioration of one or more symptoms of a disorder, or preventadvancement of a disorder, or cause regression of the disorder. Forexample, with respect to the treatment and/or prevention of plateletaggregation onto a blood vessel, in one embodiment, a therapeuticallyeffective amount will refer to the amount of a therapeutic agent (e.g.,a mixed disulfide conjugate of a thienopyridine compound) that decreasesthe reduces and/or prevents platelet aggregation by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 100%.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable vehicle” encompasses any of the standard pharmaceuticalcarriers, solvents, surfactants, or vehicles. Suitable pharmaceuticallyacceptable vehicles include aqueous vehicles and nonaqueous vehicles.Standard pharmaceutical carriers and their formulations are described inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,19th ed. 1995.

DETAILED DESCRIPTION OF THE INVENTION

Thienopyridinyl antiplatelet agents include three clinically used drugs,clopidogrel (Plavix), ticlopidine (Ticlid), and prasugrel (Effient).Their chemical structures and IUPAC names for clopidogrel (Plavix),ticlopidine (Ticlid), and prasugrel (Effient) are as follows:

(ticlopidine;5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2c]pyridine),

(clopidogrel; (+)-(S)-methyl2-(2-chlorophenyl)-2-(6,7-dihdrothieno[3,2-c]pyridine-5(4H)-yl)acetate),and

prasugrel;(RS)-5-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-ylacetate).

Thienopyridinyl antiplatelet agents are widely used to treat patientswith acute cardiovascular syndromes and peripheral vascular diseases,particularly among those undergoing percutaneous coronary intervention(e.g., coronary angioplasty) to prevent heart attack and stroke. Nearlytwo million patients receive coronary and carotid stents every year inthe United States and the annual sales for Plavix alone was worth $6.5billion in 2010.

In spite of widespread use, clopidogrel has shown significantinter-individual variability in its efficacy (see, e.g., Freedman J Eand Hylek E M (2009) New Engl J Med 360(4):411-413; Gurbel P A andTantry U S (2007) Thromb Res 120(3):311-321; Sofi F, et al., (2011)Pharmacogenomics J 11(3):199-206). Nearly one-third of patients do notrespond to clopidogrel therapy (see, e.g., Mason P J, Jacobs A K andFreedman J E (2005) J Am Coll Cardiol 46(6):986-993). A large number ofstudies have been carried out attempting to identify genetic markersthat correlate with the lack of response with the aim of overcoming thisinter-individual variability. It has been shown that clopidogrel is lesseffective in patients who carry the mutant CYP2C19*2 gene (see, e.g.,Dick R J, Dear A E and Byron K A (2011) Heart Lung Circ 20(10):657-658;Shuldiner A R, et al., (2009) JAMA 302(8):849-857; Sofi F, et al.,(2011) Pharmacogenomics J 11(3): 199-206). However the CYP2C19*2 mutantgene accounts for only 12% of the variations in response (see, e.g.,Shuldiner A R, et al., (2009) JAMA 302(8):849-857). Other factors arelikely involved, but have not been identified.

Indeed, though widely used as antiplatelet agents, there are drawbacksassociated with thienopyridinyl antiplatelet agents. A major shortcomingfor clopidogrel is a dosing inconsistency. For example, nearly one-thirdof patients do not respond to clopidogrel treatment. Ticlopidine cancause a series of adverse effects ranging from moderate symptoms of skinrashes and diarrhea to severe and sometimes fatal ones such asneutropenia and bone marrow aplasia. In rare cases it causes severeidiosyncratic events of agranulocytosis. Excessive bleeding has beenassociated with the use of prasugrel, particularly in older patients.

Such drawbacks associated with thienopyridinyl antiplatelet agents areclosely related to the fact that these three drugs are all prodrugs thatrequire oxidative bioactivation to the active metabolite (AM) bypolymorphic cytochromes P450 (P450s) as illustrated in Scheme 1. Becauseof this oxidative bioactivation process, the amount of the activemetabolite produced by P450s varies with the genetic makeup of eachpatient's hepatic P450s. Furthermore, these drugs are extensivelymetabolized by P450s to produce multiple metabolites, some of which arehighly reactive and potentially toxic. It has been reported that thesevere idiosyncratic events due to ticlopidine are associated with theproduction of reactive metabolites.

As noted, the variable response to clopidogrel therapy is closelyrelated to the fact that clopidogrel is a prodrug that requiresoxidative bioactivation by cytochromes P450 (P450s) to itspharmacologically active metabolite (AM) (see. e.g., Kazui M, et al.,(2010) Drug Metab Dispos 38(1):92-99; Savi P, et al., (2000) ThrombHaemost 84(5):891-896). It is well documented that P450-mediatedbioactivation involves two consecutive oxidative steps (see, e.g.,Dansette P M, Thebault S, Bertho G and Mansuy D (2010) Chem Res Toxicol23(7):1268-1274; Dansette P M, Rosi J, Bertho G and Mansuy D (2012) ChemRes Toxicol 25(2):348-356); clopidogrel is first monoxygenated to2-oxoclopidogrel, which is in turn oxidized to the AM in the secondstep. Although it has been argued that esterase PON1 is responsible forconverting 2-oxoclopidogrel to the AM (see, e.g., Bouman H J, et al.,(2011) Nat Med 17(1):110-116), increasing evidence supports the ideathat 2-oxoclopidogrel is converted to the AM via a sulfenic acidintermediate (see. e.g., Dansette P M, Libraire J, Bertho G and Mansuy D(2009) Chem Res Toxicol 22(2):369-373; Dansette P M, Rosi J, Bertho Gand Mansuy D (2012) Chem Res Toxicol 25(2):348-356; Dansette P M, RosiJ, Debernardi J, Bertho G and Mansuy D (2012) Chem Res Toxicol25(5):1058-1065; Dansette P M, Thebault S, Bertho G and Mansuy D (2010)Chem Res Toxicol 23(7):1268-1274), as illustrated in Scheme 2.

According to Scheme 2, 2-oxoclopidogrel is first oxidized to a sulfenicacid intermediate by P450s. The highly unstable sulfenic acid is thenrapidly reduced by glutathione (GSH) to form a mixed disulfide conjugate(RS-SG) that is subsequently reduced by another GSH molecule to form theAM. This is consistent with the observation that GSH is required for theformation of the AM in human liver microsomes (HLMs) (see, e.g., KazuiM, et al., (2010) Drug Metab Dispos 38(1):92-99). It is widely acceptedthat the AM is responsible for inhibition of platelet aggregationthrough covalent modification of platelet P2Y₁₂ receptor (see, e.g.,Ding Z, et al., (2003) Blood 101(10):3908-3914; Algaier I, et al.,(2008) J Thromb Haemost 6(11):1908-1914). The anti-platelet activity ofthe mixed disulfide conjugate RS-SG remains untested.

Metabolism of 2-oxoclopidogrel in the presence of N-acetyl-L-cysteine(NAC) and L-cysteine leads to the formation of both the AM and mixeddisulfide conjugates (see, e.g., Zhang H. Lau W C and Hollenberg P F(2012) Mol Pharmacol 82:302-309). In addition, it was demonstrated thatthe mixed disulfide conjugates of NAC and L-cysteine exchange thiolswith GSH and that the equilibrium between the AM, the AM conjugate andGSH is governed by their redox potentials. The redox potential of thesulfenic acid intermediate is likely to be high because it is a reactiveoxidant.

To overcome drawbacks associated with thienopyridine compounds, mixeddisulfide conjugates of thienopyridine compounds were developed.Experiments conducted during the course of developing embodiments forthe present invention demonstrated that the mixed disulfide conjugatesof thienopyridine compounds of the present invention are capable ofproducing active metabolites in the presence of endogenous glutathione(GSH) without the need for bioactivation by P450s, as illustrated inScheme 3. This approach not only bypasses the oxidative bioactivationprocess by P450s, but circumvents many of the drawbacks of thethienopyridinyl drugs. For example, the mixed disulfide conjugates ofthienopyridine compounds of the present invention improve dosingconsistency because production of the active metabolite from theconjugates is predictable. In addition, use of the mixed disulfideconjugates of thienopyridine compounds of the present invention asantiplatelet agents reduce the toxicity because toxic reactivemetabolites are not produced by the thiol-exchange reaction. Inaddition, the therapeutic onset time for the mixed disulfide conjugatesof thienopyridine compounds of the present invention will be shortened,which greatly benefits patients who experience acute cardiovascularevents. The standard regimen for thienopyridines requires continuouslydosing patients for 3-5 days because only a small percentage of ingestedthienopyridines are converted to the active metabolite. In contrast themixed disulfide conjugates of thienopyridine compounds of the presentinvention can release the active metabolites with high yields in lessthan 30 min. In addition, the mixed disulfide conjugates ofthienopyridine compounds of the present invention have superiorstability over the active metabolites and therefore they can be used toquantitatively generate the active metabolites for basic and clinicalresearch in vitro.

Within Scheme 3, examples of SR include, but are not limited to,

(6-chloropyridazine-3-thiol (CPT)),

3-nitropyridine-2-thiol (NPT)),

(2,5-dimethylfuran-3-thiol (DFT)),

(L-cysteine (CYS)),

(g-L-glutamyl-L-cysteine (GC)),

(Cysteine-Glycine (CG)),

(2-mercaptoethanol (BME)),

(cysteamine (CYA)),

(N-acetyl-L-cysteine (NAC)), and

(glutathione (GSH)).

Accordingly, the present invention relates to mixed disulfide conjugatesof thienopyridine compounds which are capable of producing activethienopyridine metabolites in the presence of endogenous glutathione(GSH) without the need for bioactivation by P450s. The invention furtherrelates to methods of treating, ameliorating, or preventingcardiovascular disorders in a patient, such as those that are responsiveto antiplatelet agents (such as clopidogrel, ticlopidine, and prasugrel)comprising administering to a patient a mixed disulfide conjugate of athienopyridine compound of the invention. Such disorders include, butare not limited to, coronary artery disease, peripheral vasculardisease, and cerebrovascular disease. In some embodiments, the mixeddisulfide conjugates of thienopyridine compounds are used to inhibitplatelet aggregation by, for example, altering the function of plateletmembranes by blocking ADP receptors (e.g., thereby preventing aconformational change of glycoprotein IIb/IIIa which allows plateletbinding to fibrinogen). In some embodiments, the mixed disulfideconjugates of thienopyridine compounds reduce aggregation (“clumping”)of platelets by irreversibly binding to P2Y₁₂ receptors. In someembodiments, the mixed disulfide conjugates of thienopyridine compoundsare used within pharmaceutical compostions configured for intravenous(IV) administration (e.g., in medical situations requiring IVadministration of antiplate agents (e.g., coronary angioplasty)).

The present invention is not limited to particular mixed disulfideconjugates of thienopyridine compounds. In some embodiments, the mixeddisulfide conjugates of thienopyridine compounds are described byFormula I:

including pharmaceutically acceptable salts, solvates, and/or prodrugsthereof.

Formula I is not limited to a particular chemical moiety for R1, R2, andR3. In some embodiments, R1, R2, R3 each independently include anychemical moiety that renders the resulting compound capable of producingactive thienopyridine metabolites in the presence of endogenousglutathione (GSH) without the need for bioactivation by P450s. In someembodiments, R1, R2, R3 each independently include any chemical moietythat renders the resulting compound capable of treating, ameliorating,or preventing cardiovascular disorders (e.g., coronary artery disease,peripheral vascular disease, and cerebrovascular disease) in a patient,such as those that are responsive to antiplatelet agents (such asclopidogrel, ticlopidine, and prasugrel). In some embodiments, R1, R2,R3 each independently include any chemical moiety that renders theresulting compound capable of inhibiting platelet aggregation by, forexample, altering the function of platelet membranes by blocking ADPreceptors (e.g., thereby preventing a conformational change ofglycoprotein IIb/IIIa which allows platelet binding to fibrinogen). Insome embodiments, R1, R2, R3 each independently include any chemicalmoiety that renders the resulting compound capable of reducingaggregation (“clumping”) of platelets by irreversibly binding to P2Y₁₂receptors.

In some embodiments, R1 is H, —CO—OCH3, or

In some embodiments, R3 is Chlorine or Fluorine.

In some embodiments, R2 is selected from, but not limited to,

(6-chloropyridazine-3-thiol (CPT)),

3-nitropyridine-2-thiol (NPT)),

(2,5-dimethylfuran-3-thiol (DFT)),

(L-cysteine (CYS)),

(g-L-glutamyl-L-cysteine (GC)),

(Cysteine-Glycine (CG)),

(2-mercaptoethanol (BME)),

(cysteamine (CYA)),

(N-acetyl-L-cysteine (NAC)), and

(glutathione (GSH)).

In some embodiments, the following mixed disulfide conjugates ofthienopyridine compounds are contemplated for Formula I:

In some embodiments, the following mixed disulfide conjugates ofthienopyridine compounds are contemplated for Formula I:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, the mixed disulfide conjugates of thienopyridinecompounds are used to treat, ameliorate, or prevent cardiovasculardisorders in an animal (e.g., a mammalian patient including, but notlimited to, humans and veterinary animals), such as those that areresponsive to antiplatelet agents (such as clopidogrel, ticlopidine, andprasugrel) comprising administering to a patient a mixed disulfideconjugate of thienopyridine compound of the invention. Such disordersinclude, but are not limited to, coronary artery disease, peripheralvascular disease, atherothrombosis, and cerebrovascular disease. Indeed,in some embodiments, the mixed disulfide conjugates of thienopyridinecompounds are used to decrease platelet aggregation and/or inhibitthrombus formation. In this regard, such diseases and pathologies areamenable to treatment or prophylaxis using the present methods and mixeddisulfide conjugates of thienopyridine compounds.

In some embodiments, the mixed disulfide conjugates of thienopyridinecompounds are used in the prevention of vascular ischemic events inpatients with symptomatic artherosclerosis. In some embodiments, themixed disulfide conjugates of thienopyridine compounds are used to treator prevent acute coronary syndrome without ST-segment elevation. In someembodiments, the mixed disulfide conjugates of thienopyridine compoundsare used for the prevention of thrombosis after placement ofintracoronary stent. In some embodiments, the mixed disulfide conjugatesof thienopyridine compounds are used to inhibit platelet aggregation by,for example, altering the function of platelet membranes by blocking ADPreceptors (e.g., thereby preventing a conformational change ofglycoprotein IIb/IIIa which allows platelet binding to fibrinogen). Insome embodiments, the mixed disulfide conjugates of thienopyridinecompounds reduce aggregation (“clumping”) of platelets by irreversiblybinding to P2Y₁₂ receptors. In some embodiments, the mixed disulfideconjugates of thienopyridine compounds are used to prolong bleedingtime. In some embodiments, the mixed disulfide conjugates ofthienopyridine compounds are used to decrease incidence of stroke inhigh-risk patients.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising mixed disulfide conjugates of thienopyridinecompounds configured for intravenous (IV) administration. In someembodiments, such pharmaceutical compositions comprising mixed disulfideconjugates of thienopyridine compounds configured for intravenous (IV)administration are used in the treatment, amelioration and prevention ofatherothrombosis. In some embodiments, such pharmaceutical compositionscomprising mixed disulfide conjugates of thienopyridine compoundsconfigured for intravenous (IV) administration are used for rapidinhibition of platelet aggregation. In some embodiments, suchpharmaceutical compositions comprising mixed disulfide conjugates ofthienopyridine compounds configured for intravenous (IV) administrationare used during percutaneous coronary intervention procedures (e.g.,coronary angioplasty) for rapid inhibition of platelet aggregation.Indeed, anti-platelet therapy is at the cornerstone of prevention andtreatment of atherothrombosis. Platelet activation by agonists such asplaque rupture and sheer pressure stress from stents plays an importantrole in the development of atherothrombosis. Under certain clinicalsituations where patients suffer acute cardiovascular syndromes orundergo percutaneous cardiovascular intervention, rapid and completeinhibition of platelet aggregation is needed to prevent cardiovasculardeaths and ischemic complications. Such medical scenarios requireintravenous administration of anti-platelet agents that possess shortonset time. However, this is still an unmet medical need since theanti-platelet agents currently being used either have slow onset time orcannot be administrated intravenously (see, e.g., Silvain, J., andMontalescot, G., (2012) Circ. Cariovasc. Interv. 5:328-331). The mixeddisulfide conjugates of thienopyridine compounds of the presentinvention fulfill this unmet medical need as such compounds can beadministrated both orally and intravenously and possess short onsettime.

Some embodiments of the present invention provide methods foradministering an effective amount of a mixed disulfide conjugate of athienopyridine compound of the invention and at least one additionaltherapeutic agent (including, but not limited to, a therapeutic agentknown to treat, ameliorate, or prevent cardiovascular disorders), and/ortherapeutic technique (e.g., a surgical intervention). A number oftherapeutic agents known to treat, ameliorate, or prevent cardiovasculardisorders are contemplated for use in the methods of the presentinvention. Indeed, the present invention contemplates, but is notlimited to, administration of numerous therapeutic agents known totreat, ameliorate, or prevent cardiovascular disorders. Examplesinclude, but are not limited to, HMG-CoA reductase inhibitors (e.g.,Atorvastatin (Lipitor), Pravastatin (Pravachol) Simvastatin (Zocor),Rosuvastatin (Crestor), Pitavastatin (Livalo), Lovastatin (Mevacor,Altocor), Fluvastatin (Lescol)), ACE Inhibitors (e.g., Ramipril(Altace), Quinapril (Accupril), Captopril (Capoten), Enalapril(Vasotec), Lisinopril (Zestril)). Calcium Channel Blockers (e.g.,Amlodipine (Norvasc), Nifedipine (Procardia), Verapamil (Calan),Felodipine (Plendil), Diltiazem (Cardizem)), Platelet AggregationInhibitors (other than Ticlopidine, Clopidogrel, and Prasugrel) (e.g.,Abciximab (ReoPro), Aspirin, Warfarin (Coumadin), Heparin),Polyunsaturated Fatty Acids (e.g., Omega-3 polyunsaturated fatty acid(Fish Oil)), Fibric Acid Derivatives (e.g., Fenofibrate (Tricor),Gemfibrozil (Lopid)), Bile Acid Sequestrants (e.g., Colestipol(Colestid), Cholestyramine (Questran)), Antioxidants (e.g., Vitamin E),Nicotinic Acid Derivatives (e.g., Niacin (Niaspan), Thromboytic agents(e.g., Alteplase (Activase)), and Antianginal Agents (e.g., Ranolazine(Ranexa).

In some embodiments of the present invention, a mixed disulfideconjugate of thienopyridine compound of the invention and one or moreadditional therapeutic agent is administered to an patient under one ormore of the following conditions: at different periodicities, atdifferent durations, at different concentrations, by differentadministration routes, etc. In some embodiments, the mixed disulfideconjugate of thienopyridine compound is administered prior to theadditional therapeutic agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to theadministration of the additional therapeutic agent. In some embodiments,the mixed disulfide conjugate of thienopyridine compound is administeredafter the additional therapeutic agent, e.g., 0.5, 1, 2, 3, 4, 5, 10,12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks afterthe administration of the additional therapeutic agent. In someembodiments, the mixed disulfide conjugate of thienopyridine compoundand the additional therapeutic agent are administered concurrently buton different schedules, e.g., the mixed disulfide conjugate ofthienopyridine compound is administered daily while the additionaltherapeutic agent is administered once a week, once every two weeks,once every three weeks, or once every four weeks. In other embodiments,the mixed disulfide conjugate of thienopyridine compound is administeredonce a week while the additional therapeutic agent is administereddaily, once a week, once every two weeks, once every three weeks, oronce every four weeks.

Compositions within the scope of this invention include all compositionswherein the mixed disulfide conjugates of thienopyridine compounds ofthe present invention are contained in an amount which is effective toachieve its intended purpose. While individual needs vary, determinationof optimal ranges of effective amounts of each component is within theskill of the art. Typically, the compounds may be administered tomammals, e.g. humans, orally at a dose of 0.0025 to 50 mg/kg, or anequivalent amount of the pharmaceutically acceptable salt thereof, perday of the body weight of the mammal being treated for disordersresponsive to induction of apoptosis. In one embodiment, about 0.01 toabout 25 mg/kg is orally administered to treat, ameliorate, or preventsuch disorders. For intramuscular injection, the dose is generally aboutone-half of the oral dose. For example, a suitable intramuscular dosewould be about 0.0025 to about 25 mg/kg, or from about 0.01 to about 5mg/kg.

The unit oral dose may comprise from about 0.01 to about 1000 mg, forexample, about 0.1 to about 100 mg of the mixed disulfide conjugate ofthienopyridine compound. The unit dose may be administered one or moretimes daily as one or more tablets or capsules each containing fromabout 0.1 to about 10 mg, conveniently about 0.25 to 50 mg of thecompound or its solvates.

In a topical formulation, the compound may be present at a concentrationof about 0.01 to 100 mg per gram of carrier. In a one embodiment, themixed disulfide conjugate of thienopyridine compound is present at aconcentration of about 0.07-1.0 mg/ml, for example, about 0.1-0.5 mg/ml,and in one embodiment, about 0.4 mg/ml.

In addition to administering the mixed disulfide conjugate ofthienopyridine compound as a raw chemical, the compounds of theinvention may be administered as part of a pharmaceutical preparationcontaining suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the compoundsinto preparations which can be used pharmaceutically. The preparations,particularly those preparations which can be administered orally ortopically and which can be used for one type of administration, such astablets, dragees, slow release lozenges and capsules, mouth rinses andmouth washes, gels, liquid suspensions, hair rinses, hair gels, shampoosand also preparations which can be administered rectally, such assuppositories, as well as suitable solutions for administration byintravenous infusion, injection, topically or orally, contain from about0.01 to 99 percent, in one embodiment from about 0.25 to 75 percent ofactive compound(s), together with the excipient.

The pharmaceutical compositions of the invention may be administered toany patient which may experience the beneficial effects of the mixeddisulfide conjugates of thienopyridine compounds of the invention.Foremost among such patients are mammals, e.g., humans, although theinvention is not intended to be so limited. Other patients includeveterinary animals (cows, sheep, pigs, horses, dogs, cats and the like).

The compounds and pharmaceutical compositions thereof may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions maybe used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs orpigments may be added to the tablets or dragee coatings, for example,for identification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are in oneembodiment dissolved or suspended in suitable liquids, such as fattyoils, or liquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

The topical compositions of this invention are formulated in oneembodiment as oils, creams, lotions, ointments and the like by choice ofappropriate carriers. Suitable carriers include vegetable or mineraloils, white petrolatum (white soft paraffin), branched chain fats oroils, animal fats and high molecular weight alcohol (greater than C₁₂).The carriers may be those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers can be employed in thesetopical formulations. Examples of such enhancers can be found in U.S.Pat. Nos. 3,989,816 and 4,444,762.

Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil such as almond oil with warm soft paraffinand allowing the mixture to cool. A typical example of such an ointmentis one which includes about 30% almond oil and about 70% white softparaffin by weight. Lotions may be conveniently prepared by dissolvingthe active ingredient, in a suitable high molecular weight alcohol suchas propylene glycol or polyethylene glycol.

One of ordinary skill in the art will readily recognize that theforegoing represents merely a detailed description of certain preferredembodiments of the present invention. Various modifications andalterations of the compositions and methods described above can readilybe achieved using expertise available in the art and are within thescope of the invention.

EXAMPLES

The following examples are illustrative, but not limiting, of thecompounds, compositions, and methods of the present invention. Othersuitable modifications and adaptations of the variety of conditions andparameters normally encountered in clinical therapy and which areobvious to those skilled in the art are within the spirit and scope ofthe invention.

Example I

This example describes the synthesis of mixed disulfide conjugates ofclopidogrel and ticlopidine.

Synthesis of mixed disulfide conjugates of clopidogrel and ticlopidinewas carried out in 50 mM potassium phosphate buffer using human livermicrosomes (HLMs) according to Scheme 4.

Within Scheme 4, RS (or —SR) are thiol-containing reagents selectedfrom, but not limited to,

(6-chloropyridazine-3-thiol (CPT)),

3-nitropyridine-2-thiol (NPT)),

(2,5-dimethylfuran-3-thiol (DFT)),

(L-cysteine (CYS)),

(g-L-glutamyl-L-cysteine (GC)),

(Cysteine-Glycine (CG)),

(2-mercaptoethanol BME)),

(cysteamine (CYA)),

(N-acetyl-L-cysteine (NAC)), and

(glutathione (GSH)). Within Scheme 4, AM-SR represents a mixed disulfideconjugate of the present invention.

The results showed that all of the ten RS compounds formed therespective conjugates. In addition, it was confirmed that the reactantRS forms conjugate with the active metabolite through mixed disulfidebonds using tandem mass spectrometry. The conjugates were purified fromthe reaction mixtures using reverse phase chromatography.

Example II

This example describes production of the active metabolites from themixed disulfide conjugate compounds.

The Conjugates Clop-CPT

(Z)-2-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4-((6-chloropyridazin-3-yl)disulfanyl)piperidin-3-ylidene)aceticacid] and Tic-NPT

(Z)-2-(1-(2-chlorobenzyl)-4-((3-nitropyridin-2-yl)disulfanyl)piperidin-3-ylidene)aceticacid] were chosen for further studies.

The ability of the mixed disulfide conjugate Clop-CPT and Tic-NPT toproduce the active metabolites (AM) in the presence of glutathione wasnext tested. Clop-CPT was rapidly reduced by 1 mM GSH with a concomitantincrease in the amount of the AM of clopidogrel. The half-life for theproduction of the AM of clopidogrel from the clop-CPT conjugate was only1.8 min. The same is true for tic-NPT conjugate, but the half-life was14.7 min.

Example III

This example describes inhibition of platelet aggregation by mixeddisulfide conjugates of thienopyridine compounds of the presentinvention.

To demonstrate the mixed disulfide conjugates of thienopyridinecompounds of the present invention are capable of inhibiting plateletaggregation in the presence of GSH, platelet aggregation assays wereconducted. Approximately 20 ml blood was drawn from rabbits andplatelets were collected by centrifugation whereas the supernatants werecollected as platelet poor plasma (PPP). Prior to the inhibition assay,the conjugates of Clop-CPT and Tic-NPT were dissolved in 0.5 ml PPP andincubated with 1 mM GSH at 37° C. for 30 min to produce the activemetabolite. The platelets were then re-suspended gently in the PPPcontaining the active metabolite. After incubation at 37° C. for onehour, platelet aggregation was initiated by the addition of 5 μM of theagonist ADP. Platelet aggregation was then recorded using anaggregometer.

Conjugates of Clop-CPT and Tic-NPT inhibited platelet aggregation byapproximately 60% in the presence of 1 mM GSH compared with the negativecontrol that contained no conjugate. This level of inhibition wasapproximately the same as the positive control that contained the activemetabolite generated from human liver microsomes (HLMs).

Example IV

This example describes the materials and methods for Examples 5-9.

Chemicals.

(S)-clopidogrel, racemic 2-oxoclopidogrel, and cis-clopidogrel-MP werepurchased from Toronto Research Company (Ontario, Canada). Glutathione(GSH), γ-L-glutamyl-L-cysteine (GC), Cys-Gly (CG), L-cysteine,β-mercaptoethanol (BME), N-acetyl-L-cysteine (NAC), cysteamine (CYA)hydrochloride, 2,5-dimethylfuran-3-thiol, 6-chloropyridazine-3-thiol,3-nitropyridine-2-thiol, and 2-bromo-3′-methoxyacetophenone (MPB) werepurchased from Sigma-Aldrich company (St. Louis, Mo.). Pooled HLMs andcytosol were purchased from XenoTech (Lenexa, Kans.).

Determination of the Rate for the Formation of the AM by HLMs in thePresence of Various Thiol Reductants.

To examine the effects of various thiol reductants on the formation ofthe active metabolite (AM), the rates at which the AM was produced weredetermined. Production of the AM was performed in 0.1 ml of 50 mMpotassium phosphate (KPi) buffer (pH 7.4) containing 0.2 mg/mL HLM, 0.1mM 2-oxoclopidogrel, the NADPH-regenerating system, and 1 mM of each ofthe thiol reductants except that 0.3 mM CPT, DFT, or NPT were used. Thereaction was initiated by the addition of 5 units of glucose-6-phosphatedehydrogenase (G6PD) and incubated at 37° C. for 20 min. The AM was thenderivatized with 4 mM MPB at room temperature for 10 min, followed byacidification with acetic acid to 3% (v/v). For quantification, 50pmoles of (S)-clopidogrel was added into each reaction mixture asinternal standard (1S). The derivatized AM (AM-MP) was quantitated usingLC-MS/MS.

The MS analyses of the reaction mixture were performed on an ion-trapmass spectrometer (LCQ DecaXP, Thermo Fisher Scientific, Waltham, Mass.)as reported previously (Zhang et al., 2012). In brief, the metabolitesof 2-oxoclopidogrel were separated on a reverse phase C18 column (2×100mm, 3 μm, Phenemonex, CA) using a binary mobile phase at a flow rate of0.2 ml/min. The temperature of the C18 column was maintained at 40° C.using a column heater (Restek Corporation, Lancaster, Pa.). The massspectrometer was operated in positive electrospray ionization mode withthe following settings: heated capillary temperature, 200° C.; Sprayvoltage, +4.5 kV; sheath gas flow, 60 (arbitrary units); auxiliary gas,20 (arbitrary units). The AM-MP and IS were fragmented in the MS throughcollision-induced dissociation (CID) at 35% energy level. Transitionsfrom m/z 504→m/z 354 for the AM-MP and from m/z 322→m/z 212 for the ISwere used to quantitate the amount of the AM-MP based on a calibrationcurve consisting of various concentrations of cis-clopidogrel-MP.

Analyses of the Mixed Disulfide Conjugates of Clopidogrel UsingLC-MS/MS.

The mixed disulfide conjugates were produced by HLMs for both structuraland semi-quantitative analyses. Metabolism of 2-oxoclopidogrel wasperformed in 0.2 ml of 50 mM KP buffer (pH 7.4) as described aboveexcept that the concentration of HLMs was increased to 1 mg/ml. Thereaction was incubated at 37° C. for 30 min and then quenched by theaddition of 0.1 ml of 10% of acetic acid in acetonitrile. The quenchedsamples were centrifuged at 13,000×g for 10 min to remove the HLMs.Aliquots of 50 μl of the supernatant were loaded onto a massspectrometer to analyze both the active and conjugate metabolites.

The MS analyses were performed as described above except that the MSdetector was operated in the dependent scan mode. The precursor ionswere scanned from m/z 300-700, whereas the MS² spectra were obtainedfrom m/z 100 to 700 for the four most abundant ions. Forsemi-quantitative analysis, 50 pmoles of the IS was spiked into eachsample that had been quenched. The relative amounts of the AM andrespective conjugates were calculated as the AUC ratios of themetabolites to that of the IS.

Determination of the Kinetics for the Conversion of the Mixed DisulfideConjugates to the AM.

To examine the reactivity of the mixed disulfide conjugates, thekinetics for the reduction of the mixed disulfide conjugates by GSH weredetermined. The mixed disulfide conjugates were generated in 1 ml of 50mM KPi buffer (pH 7.4) buffer containing 1 mg/ml HLM, 0.1 mM2-oxoclopidogrel and 0.3 or 1 mM thiol reductants. The reaction wasincubated at 37° C. for 50 min after initiated by the addition of G6PD.The reaction mixture was then centrifuged at 13,000×g to remove the HLMsand the supernatant was loaded to a pre-conditioned SPE cartridge (C18,100 mg/1 ml, Agilent Technologies, CA) and the mixed disulfide conjugatewas eluted with 2 ml of methanol. The eluent was then dried using aSpeedvac concentrator and the dried sample was stored at −80° C. untiluse. Prior to the kinetic measurements, the dried samples were firstre-dissolved in 0.5 ml of 50 mM KPi buffer (pH 7.4) and thenequilibrated at 37° C. for 5 min. Small aliquots (1-5 μl) of stock GSHand cytosol (when present) solutions were added to the conjugate samplesat 1 mM and 0.2 mg/ml, respectively, to initiate the thiol-disulfideexchange reaction. At designated times, an aliquot of 50 μl of thereaction mixture was withdrawn and mixed with 25 μl of 10% acetic acidin acetonitrile to terminate the thiol-disulfide exchange reaction. Thet=0 sample was prepared immediately prior to the addition of GSH. Theamounts of the mixed disulfide conjugates and the AM were analyzed usingLC-MS/MS as described above.

Formation of Active Metabolite H4 from the Mixed Disulfide Conjugates ofClopidogrel.

Since the anti-platelet activity of the AM is closely related to itsstereochemistry, the stereochemistry of the mixed disulfide conjugatesformed in the presence of CPT. DFT or NPT was investigated because oftheir relatively high redox potentials. Due to lack of genuine standardsfor the stereoisomers of the AM, the mixed disulfide conjugates with GSHwere first treated to release the AM and then derivatized the AM withMPB so as to compare the AM-MP derivatives with the cis-clopidogrel-MPstandard. Preparation and reduction of the conjugates with GSH wereperformed as described above. After an incubation of 20 min at 37° C.with 1 mM GSH, MPB was added at 4 mM to alkylate the AM. The alkylationreaction was terminated in 10 min by the addition of half a volume of10% acetic acid in acetonitrile. An aliquot of 50 μl of the reactionmixture was subjected to LC-MS/MS analysis as described for thequantitation of the AM.

Anti-Platelet Activity of the Mixed Disulfide Conjugates of Clopidogrel.

In order to generate sufficient quantities of the mixed disulfideconjugates, the metabolism of 2-oxoclopidogrel was performed in 2-mlreaction mixtures containing 1 mg/ml HLM, 0.1 mM 2-oxoclopidogrel, theNADPH-regenerating system, and 0.3 mM CPT or NPT or 1 mM GSH. Thereaction was initiated by the addition of 10 units G6PD and incubated at37° C. for 50 min. Two control samples were prepared in parallel. Onecontrol sample did not contain any G6PD (-G6PD), which was designed toexamine whether 2-oxoclopidogrel and other components present in theHLMs contributed to anti-platelet activities. The other control (—SH)did not contain any thiol reductant, which was intended to examinewhether any metabolites other than the AM and the conjugate interferedwith the anti-platelet activity assay. After an incubation of 50 min,the reaction mixtures were centrifuged at 13,000×g to remove the HLMs.The supernatants were loaded onto SPE C18 cartridges to enrich the mixeddisulfide conjugates. After extensive washing with water to remove saltsand other water-soluble metabolites, the conjugate samples were elutedwith 2 ml of methanol. The methanolic fractions were dried using aSpeedvac concentrator and the dried samples were then re-suspended in 1ml of platelet-poor plasma (PPP). Prior to the anti-platelet activityassays, the re-suspended conjugates were divided into two equal volumes(0.5 ml each), one of which was treated with 1 mM GSH at 37° C. for 30min to generate the AM. Both samples were then placed on ice until use.

The procedures used to determine ex vivo anti-platelet activity werepreviously reported (see, e.g., Abell L M and Liu E C (2011) J Pharm ExpTher 339(2):589-596).

Male New Zealand white rabbits (2.2-2.9 kg) were used as blood donors.Whole blood was drawn from a central ear artery into a plastic syringecontaining 3.7% sodium citrate as the anticoagulant (1:10 volume ratioof citrate to blood). A whole blood cell count was determined with aMedonic CA620 hematology analyzer (Clinical Diagnostic Solutions, Inc.,Plantation, Fla., USA). Platelet-rich plasma (PRP), the supernatantpresent after centrifugation of whole blood at 100×g for 10 min, wasdiluted with PPP to achieve a platelet count of approximately300,000/μl. Platelet-poor plasma was prepared by centrifuging theremaining blood at 1,500×g for 10 min and discarding the bottom cellularlayer. The diluted PRP was divided into 0.5 ml samples, centrifugedagain at 170×g for 10 mins, and the resulting supernatant was discarded.The platelet pellets were re-suspended in platelet-poor plasmacontaining the various chemical inhibitors prepared as describedpreviously and incubated with gentle shaking at 37° C. for 60 min tomodify the P2Y₁₂ receptor. Ex vivo platelet aggregation was assessed byestablished nephelometric methods with the use of a 4-channelaggregometer (BioData PAP-4; BioData Corp., Horsham, Pa. USA) byrecording the increase in light transmission through a stirredsuspension of PRP maintained at 37° C. Platelet aggregation was inducedwith ADP (10 M). A subaggregatory concentration of epinephrine (550 nM)was used to prime the platelets before addition of the agonist.

Example V

This example describes the effects of thiol reductants on the formationof the active metabolite (AM) of clopidogrel. To examine the effects ofthiol reductants, the steady-state rates for the formation of the AM inthe presence of various thiol reductants was determined. Theconcentrations of the thiol reductants present in the metabolicreactions were 1 mM except for CPT, DPT and NPT. Instead, theconcentrations of these three thiol reductants were 0.3 mM because oftheir low K_(m) values. As shown in FIG. 1, the AM is formed in thepresence of all but three thiol reductants. The highest rate for theformation of the AM was observed in the presence of GSH, an endogenousreductant in the human body. Specifically, in the presence of 1 mM GSH,the AM is produced at a rate of 167 pmole AM/min/mg HLM. Likewise,L-cysteine is ˜84% as active as GSH in producing the AM. As observedpreviously (see, e.g., Zhang H, et al., (2012) Mol Pharmacol82:302-309), only a low level of the AM was formed in the presence of 1mM NAC. The rate is only ˜7% of that observed in the presence of 1 mMGSH. No AM was observed in the presence of CPT, DFT, and NPT. Overallthe rates for the formation of the AM decrease in the order ofGSH>CYS>CG>GC>CYA>BME>NAC>CPT or DFT or NPT. This wide range of ratesunderscores the critical role of thiol reductants in the formation ofthe AM.

Example VI

This example describes analyses of the mixed disulfide conjugates ofclopidogrel. Formation of the AM was greatly affected by the thiolreductants present. In this experiment, the effects of the thiolreductants on the formation of the mixed disulfide conjugates wasexamined. This is particularly important in order to understand thecause for the lack of any AM formed in the presence of CPT, DFT, andNPT. In marked contrast to what was observed for the AM, mixed disulfideconjugates were formed in the presence of all the thiol reductantsexamined. The m/z for the parent ions MH⁺ and the retention times ofthese mixed disulfide conjugates are summarized in Table 1, and theextracted ion chromatograms (EICs) of four selected mixed disulfideconjugates are presented in FIG. 2. The parent ions MH⁺ observed are inexcellent agreement with the theoretical values for these conjugates. Inthe presence of β-mercaptoethanol (BME), four conjugate peaks wereobserved at m/z 432 eluting from 8.9 to 9.72 min (FIG. 2A). These fourAM peaks are likely due to the formation of multiple stereoisomers ofclopidogrel as reported previously (see, e.g., Pereillo J M, et al.(2002) Drug Metab Dispos 30(11):1288-1295). Two major conjugate peakswere observed in the presence of CPT and NPT (FIGS. 2C and 4D,respectively). However, in the presence of DFT, one predominantconjugate peak with m/z 482 was observed at 15.8 min (FIG. 2B). TheK_(m) values for the formation of the CPT, NPT and DFT conjugates weredetermined to be 23, 51 and 30 μM, respectively, which is significantlylower than a K_(m) of 300 μM for GSH that we previously reported (see,e.g., Zhang H, et al., (2012) Mol Pharmacol 82:302-309).

TABLE 1 Parent ions (MH⁺) and retention times (RT) observed for themixed disulfide conjugates of clopidogrel in LC-MS analysis. MH⁺ RT^(a)MH⁺ Theoretical^(b) Thiol Compounds Abbreviation (m/z) (min) (m/z)Glutathione GSH 661.05 5.47 661.02 L-cysteine CYS 475.05^(c) 5.60^(c)475.06 L-cysteine-L-glyceine CG 532.09 5.44 532.10 γ-L-glatamyl-L- GC604.09 5.71 604.12 cysteine cysteamine CYA 431.05 6.33 431.09β-mercaptoethanol BME 432.06 9.65 432.07 N-acetyl-L-cysteine NAC 517.116.85 517.09 6-chloropyridazine-3- CPT 499.99 11.97 500.03 thiol2,5-dimethylfuran-3- DFT 482.08 15.80 482.09 thiol3-nitropyridine-2-thiol NPT 510.08 12.82 510.06 ^(a)retention time forthe most intense peak; ^(b)exact masses calculated from molecularformula; ^(c)data from Zhang H, et al., (2012) Mol Pharmacol 82:302-309.

Integration of the area under the curve (AUC) for each EIC of theconjugates gave the relative amount of the mixed disulfide conjugatesproduced. As shown in FIG. 3, the relative amounts of the mixeddisulfide conjugates varied substantially from each other. Only a lowlevel of the glutathionyl conjugate was formed, indicating thatmetabolism of 2-oxoclopidogrel in the presence of GSH greatly favors theformation of the AM. Although both the AM and the mixed disulfideconjugate were formed in the presence of BME, it is clear that formationof the conjugate is favored over the AM. In spite of lack of formationof the AM, the mixed disulfide conjugates of CPT, DFT and NPT wereformed in significant quantities. Due to lack of genuine standards ofthese conjugates it was unable to quantitate the absolute amounts ofthese conjugates. Caution should be exercised in comparing the absoluteamounts of the conjugates based on the AUC ratios because theseconjugates may respond differently to the MS detector.

To determine the chemical structure of these conjugates, the MS and MS²spectra were obtained. The MS spectra of all ten conjugates showed themajor MH⁺ peaks at expected m/z ratios as summarized in Table 1, alongwith a pair of ³⁵Cl/³⁷Cl isotope peaks that is characteristic of thepresence of one Cl atom in clopidogrel. The only exception to this isthe CPT conjugate that contains two chlorine atoms. Its MS and MS²spectra are shown in FIG. 4. This conjugate was observed at m/z 499.99,which is within an experimental error of 80 ppm of the expected m/zvalue of 500.03. In addition, a strong isotope peak was also observed at501.94 with ˜75% of the intensity of the base peak, indicative of thepresence of two chlorine atoms in this conjugate. The MS² of the parention at m/z 499.99 showed the formation of multiple daughter ions. Thepredominant daughter ion was observed at m/z 353.99 with other minorones at m/z 465.90, 211.93 and 183.34. This fragmentation pattern, inaddition to the presence of the ³⁵Cl/³⁷Cl isotope peaks, is consistentwith the chemical structure of the conjugate possessing a mixeddisulfide bond shown in FIG. 4D. The predominant daughter ion m/z 353.99is assigned to the larger fragment cleaved at the mixed disulfide bondas we reported previously for the conjugates of GSH, NAC and L-cysteinewith clopidogrel (see, e.g., Zhang H, et al., (2012) Mol Pharmacol82:302-309). The daughter ions at m/z 212 and 183 are alsocharacteristic of clopidogrel (see, e.g., Dansette P M, et al., (2010)Chem Res Toxicol 23(7):1268-1274; Pereillo J M, et al., (2002) DrugMetab Dispos 30(11):1288-1295). The MS² spectrum of the isotope peak atm/z 501.94 provides further evidence for this assignment. As shown inFIG. 4C, a pair of daughter ions, instead of single ions, were observedtwo mass units apart, which support the presence of two chlorine atoms.The MS² spectra for the rest of the conjugates exhibited very similarfragmentation patterns with the characteristic daughter ions at m/z 354.

Example VII

This example describes the kinetics for the reduction of the mixeddisulfide conjugates of clopidogrel by GSH. The kinetics for thethiol-disulfide exchange reaction between the various mixed disulfideconjugates and GSH were determined and the results are shown in FIG. 5.Incubation of the conjugates with GSH and cytosol led to time-dependentdecreases in the amounts of the mixed disulfide conjugates (FIG. 5A).Fitting the kinetic data to a mono-exponential function gave first orderrate constants of 0.07, 0.79, 0.43, 1.65 and 0.13 min⁻¹ for the lossesof the mixed disulfide conjugates of BME, CPT, NAC, DFT and NPT,respectively. Since these kinetics were determined under pseudo firstorder conditions with excess of GSH (1 mM), these rate constants areequivalent to the second order rate constants of 1.2, 13, 7.2, 28 and2.2 M⁻¹s⁻¹, respectively. The data also demonstrated the variablereactivity of these conjugates toward GSH. The DFT and CPT conjugatesare 10 to 20-fold more reactive than the BME conjugate, respectively. Itappears, for example, that ˜50% of the BME conjugate still remained evenafter an incubation of 40 min, indicating, for example, that thethiol-disulfide exchange for this conjugate had reached equilibrium.

To examine the effect of cytosol and to monitor the conjugates and theAM simultaneously, the kinetics for both the formation of the AM and thereduction of the CPT conjugate in the presence and absence of cytosolwas determined. As shown in FIG. 5B, the decrease in the amount of theCPT conjugate occurred with concomitant increase in the amount of the AMwith almost identical rate constants. In the presence of cytosol, therate constants for the reduction of the conjugate and for the formationof the AM are 0.73 and 0.50 min⁻¹ respectively. In the absence ofcytosol, the reduction of the CPT conjugate is approximately one-half asfast with a rate constant of 0.39 min⁻¹ for the reduction of theconjugate and 0.35 min⁻¹ for the formation of the AM. This indicatesthat the cytosol accelerates the reduction of the thiol-disulfideexchange reaction as observed previously (see, e.g., Hagihara K, et al.,(2012) Drug Metab Dispos 40(9): 1854-1859; Hagihara K, et al., (2011)Drug Metab Dispos 39(2):208-214).

Example VIII

This example describes formation of active metabolite H4 from the mixeddisulfide conjugates of clopidogrel. As shown in Scheme 2, the activemetabolite contains two chiral centers (C7 and C4) and one double bond(C3-C16). Therefore, metabolism of racemic 2-oxoclopidogrel couldpotentially produce up to eight stereoisomers. However, only four of thediastereomers, historically referred to as H1, H2, H3 and H4, can beseparated on conventional reverse phase C18 columns, whereas the otherfour stereoisomers co-elute as enantiomers. It has been established thatH4 is responsible for the anti-platelet activity in humans and that thedouble bond of H4 is in cis configuration (see, e.g., Pereillo J M, etal., (2002) Drug Metab Dispos 30(11):1288-1295; Savi P, et al., (2000)Thromb Haemost 84(5):891-896: Tuffal G, et al., (2011) Thromb Haemost105(4):696-705). To evaluate the therapeutic potential of theseconjugates, whether H4 is formed in the mixed disulfide conjugates wasexamined and the results presented in FIG. 6.

The metabolism of 2-oxoclopidogrel by HLMs in the presence of GSH led tothe formation of four stereoisomers eluting between 8 to 11 min (dashedline, FIG. 6B). Based on the order of elution of the AM-MP derivativeson reverse phase C18 columns (see, e.g., Tuffal G, et al., (2011) ThrombHaemost 105(4):696-705), it is likely that the isomer eluting at 10.2min is the cis isomer of H4. This is consistent with the retention timeof the cis-clopidogrel-MP standard shown in FIG. 6A (solid line).Likewise the MP derivatives of the DFT conjugate also exhibited fourpeaks, similar to those observed in the presence of GSH, indicating thatthe DFT conjugate produced the cis isomers of the AM following thethiol-exchange reaction. In contrast, the MP derivative of the CPT andNPT conjugates showed two major peaks at 9.4 and 10.2 min, which isconsistent with the selective formation of the active isomer H4 (FIGS.6C & D).

Example IX

This example describes anti-platelet activity of the mixed disulfideconjugates of CPT and NPT. As a proof of concept, the anti-plateletactivities of two of the mixed disulfide conjugates, the CPT and NPTconjugates, were examined for several reasons. First, both conjugatescan be generated without the formation of any AM, which eliminates anyinterference from the AM during the anti-platelet activity assays.Second, both conjugates exchange thiols with GSH at relatively fastrates, which avoids potential decay of the AM. Third, reduction of thetwo conjugates produces the H4 isomer that is known responsible for theanti-platelet activity in humans. The results for the ex vivoanti-platelet activity assays are shown in FIG. 7. The percentage ofaggregation was normalized to that of PRP to compensate for anyvariations due to environmental factors such as blood sources, PRPpreparations, etc. As shown, the three control samples showed noinhibition of the platelet aggregation. The first control showed thatfree GSH has no effect on platelet aggregation at 1 mM concentration(GSH, FIG. 7), and the second control showed that non-metabolitecomponents present in the reaction mixtures such as 2-oxoclopidogrel andrelated impurities did not inhibit platelet aggregation (-G6PD, FIG. 7).It is known that clopidogrel may decompose to byproducts vianon-enzymatic oxidation (see, e.g., Mohan A, et al., (2008) J PharmBiomed Anal 47(1):183-189; Fayed A S, et al., (2009) J Pharm Biomed Anal49(2):193-200). These byproducts do not seem to have any inhibitoryeffects on platelet aggregation. In the third control it wasdemonstrated that the metabolites from the reaction mixture in theabsence of any thiol reductants have no effects on platelet aggregationeither. However, ˜60% inhibition of platelet aggregation in the sampleprepared from the reaction mixture containing GSH was observed (AM, FIG.7). This is expected since metabolism of 2-oxoclopidogrel in thepresence of 1 mM GSH generates the AM as shown in FIG. 1. Incubation ofPRP with the CPT and NPT conjugates did not inhibit plateletaggregation, indicating that the conjugates themselves have noanti-platelet activity (CPT & NPT, FIG. 7). In marked contrast,incubation of PRP with the CPT and NPT conjugates that had been treatedwith 1 mM GSH significantly inhibited platelet aggregation by ˜50 and70%, respectively. This inhibitory activity most likely arises from theAM released from the conjugates by GSH. These results demonstrate thatthe conjugates of clopidogrel have no anti-platelet activity and alsoconfirmed that the AM is solely responsible for the inhibition ofplatelet aggregation. Furthermore, they demonstrate that it is possibleto deliver the AM without the need for bioactivation by polymorphicP450s. It is noteworthy to point out that the variations in thepercentage of aggregation observed in the GSH, CPT+GSH, and NPT+GSHsamples are most likely due to variations in the concentrations of theAM. It was estimated that the concentrations of the AM in these sampleswere in the range of 1-4 μM.

Example X

This example describes in vivo antiplatelet activity of mixed disulfideconjugates of clopidrogrel. The antiplatelet activity of the mixeddisulfide conjugates of clopidogrel was determined in male New Zealand(NZ) white rabbits through intravenous injection. The mixed disulfideconjugates were bio-synthesized using the following technique.

Part I. Bio-Synthesis of Mixed Disulfide Conjugates of Clopidogrel

Mixed disulfide conjugates of clopidogrel were synthesized in areconstituted system containing recombinant cytochrome P450 2C19(CYP2C19) and other essential components. CYP2C19 converted2-oxoclopidogrel to a mixed disulfide conjugate in the presence ofrespective thiol compound in reconstituted systems. Scheme 5 illustratesthe bio-synthesis of the mixed disulfide conjugate between clopidogreland 3-nitropyridine-2-thiol, referred to as clopNPT.

In a typical reaction, 50 nmoles of CYP2C19, 150 nmoles of P450reductase and 250 nmoles of cytochrome b5 were reconstituted inphospholipid vesicles to form active protein complexes. 2-oxoclopidogreland 3-nitropyrine-2-thiol were added at final concentrations of 0.05 and0.3 mM respectively. Bio-synthesis of clopNPT was initiated by theaddition of 1 mM NADPH. The reaction was incubated at 37° C. for 2hours.

To purify clopNPT, the reaction mixture containing clopNPT was firstfiltered through a membrane with a cutoff of 10 kDa to remove allprotein components. The filtrate containing clopNPT was then enriched onsolid phase extraction (SPE) cartridges. ClopNPT was eluted from the SPEcartridges with 80% methanol/20% water. The eluent was concentrated to 5ml at 50° C. under vacuum. The concentrated mixture was loaded on apreparative reverse phase C18 column and ClopNPT was purified using highpressure liquid chromatography (HPLC). ClopNPT was eluted from thepreparative C18 column at a flow rate of 3 ml/min with isocratic mobilephase consisting of 42% methanol/35% acetonitrile/22.9% water/0.1%formic acid. The HPLC fractions containing clopNPT were pooled and driedunder vacuum. The final yield was ˜25%. The purity of clopNPT estimatedwith liquid chromatography-tandem mass spectrometry (LC-MS/MS) was >90%as shown in FIG. 8.

Part II. Anti-Platelet Activity of Mixed Disulfide Conjugates ofClopidogrel

The antiplatelet activity of the mixed disulfide conjugates ofclopidogrel was determined using male NZ white rabbits (1.2-1.25 kg).

To prepare intravenous solution, (S)-clopNPT was dissolved at 0.7 mg/mlin a mixture of N,N-dimethylacetamide (DMA), polyethylene glycol (PEG)400, and saline at 5, 15 and 80 (v/v) ratio. The male NZ white rabbitswere dosed at 2 mg/kg of (S)-clopNPT using two different methods. InMethod 1, the intravenous solution was first mixed with 5 mMglutathione. After an incubation of 15 min at 37° C. to activate(S)-clopNPT, the mixture was intravenously injected to the rabbit viathe jugular vein. In Method 2, the male NZ white rabbit was fed with 5ml of Readisorb glutathione solution once daily for three days toincrease cellular glutathione concentrations. On the third day,(S)-clopNPT dissolved in DMA/PEG400/saline was injected intravenously tothe Readisorb-treated rabbit. As a negative control, (S)-clopidogrel wasdosed intravenously to a male NZ white rabbit at 2 mg/kg as well. Priorto and 1 and 2 hours after dosing (S)-clopNPT, whole blood was drawnfrom the carotid artery into a plastic syringe containing 3.7% sodiumcitrate as the anticoagulant (1:10 volume ratio of citrate to blood). Awhole blood cell count was determined with a Medonic CA620 hematologyanalyzer (Clinical Diagnostic Solutions, Inc., Plantation, Fla., USA).Platelet-rich plasma (PRP), the supernatant present after centrifugationof whole blood at 100×g for 10 min, was diluted with platelet-poorplasma (PPP) to achieve a platelet count of approximately 300,000/1.Platelet-poor plasma was prepared by centrifuging the remaining blood at1,500×g for 10 min and discarding the bottom cellular layer. The dilutedPRP was divided into 0.5 ml samples, centrifuged again at 170×g for 10mins, and the resulting supernatant was discarded. Platelet aggregationwas assessed by established nephelometric methods with the use of a4-channel aggregometer (BioData PAP-4; BioData Corp., Horsham, Pa., USA)by recording the increase in light transmission through a stirredsuspension of PRP maintained at 37° C. Platelet aggregation was inducedwith ADP (10 μM). A subaggregatory concentration of epinephrine (550 nM)was used to prime the platelets before addition of the agonist.

The results are presented in FIG. 9. As shown, (S)-clopidogrel did notinhibit platelet aggregation at a dose of 2 mg/kg. However, (S)-clopNPTstrongly inhibited platelet aggregation since more than 50% of theplatelet aggregation was inhibited by (S)-clopNPT regardless whether(S)-clopNPT was dosed with glutathione or Readisorb glutathione. Theseresults demonstrated that (S)-clopNPT conjugate can be activated eitherby extraneous glutathione or endogenous glutathione. It is evident thatendogenous glutathione is more effective in activating (S)-clopNPTbecause the platelet activity of the Readisorb-treated rabbit isinhibited by ˜70% within one hour of IV injection. Numerous studies haveshown the beneficial effects of glutathione in heart disease and stroke,anti-oxidative stress, aging, etc. Co-administration of glutathione andthe mixed disulfide conjugates of clopidogrel likely offers not only thebenefit of (S)-clopNPT as antiplatelet agents, but also the benefitsassociated with the use of glutathione alone.

These results demonstrate that the mixed disulfide conjugates ofclopidogrel are more effective antiplatelet agents than clopidogrel.

Having now fully described the invention, it will be understood by thoseof skill in the art that the same can be performed within a wide andequivalent range of conditions, formulations, and other parameterswithout affecting the scope of the invention or any embodiment thereof.All patents, patent applications and publications cited herein are fullyincorporated by reference herein in their entirety.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A compound having Formula I:

or a pharmaceutically acceptable salt or solvate thereof; wherein R1 isselected from the group consisting of H, —CO—OCH3, and

wherein R2 is selected from the group consisting of

wherein R3 is Chlorine or Fluorine.
 2. A pharmaceutical compositioncomprising a compound of claim 1 and a pharmaceutically acceptablecarrier.
 3. The pharmaceutical composition of claim 2, wherein saidpharmaceutical composition is configured for intravenous administration.4. The compound of claim 1, wherein said compound is selected from thegroup consisting of


5. The compound of claim 1, wherein said compound is selected from thegroup consisting of: